Characteristics and matching of SARS-CoV-2 convalescent individuals
The study included serum samples from 37 non-hospitalized, unvaccinated convalescents (21 female, 16 male, median age: 33 years, range: 4-81) after primary infection with the Omicron variant. In all 37 individuals, a positive RT-PCR result from a nasopharyngeal swab preceded the acquisition of the respective serum sample (median interval between RT-PCR positivity and acquisition of the serum sample: 33 days, range: 16-96). The swabs were obtained during a period (January – March 2022) when the Omicron sub-lineages BA.1 or BA.2 circulated in Austria with over 98% predominance18. Furthermore, the samples of these individuals displayed significantly higher BA.1- or BA.2-specific titers of neutralizing antibodies (nAbs) than against a WT strain with the D614G mutation (B.1.1) and the Delta variant of concern (VOC) in live-virus NTs, as demonstrated previously (Supplementary Figure 1a)9.
Serum samples from 43 non-hospitalized convalescents after infection with WT virus early in the pandemic (before the emergence of VOCs, February 2020 to December 2020) served as controls. RT-PCR-positivity in controls preceded the collection of the respective serum samples with a median interval of 35 days (range: 16-70). Samples from WT controls were matched to those from convalescents after Omicron infection based on the concentration of variant-specific nAbs, age, the interval between PCR diagnosis serum sampling, and the absence of hospitalization.
Supplementary Figures 1a and b show that the matched groups of convalescents after Omicron and WT infections exhibited comparable virus-specific neutralization titers (BA.1/BA.2 vs. WT titers: p = 0.42, two-tailed Mann-Whitney U test; Supplementary Figure 1b). In addition, there was no difference in age (p = 0.32; Supplementary Figure 1c) or the interval between PCR-positivity and the time point when serum samples were obtained (p = 0.86, two-tailed Mann-Whitney U test, respectively; Supplementary Figure 1d).
Detection rates of commercial antibody assays in convalescents after Omicron infection
Serum samples from matched groups of convalescents after primary Omicron (n = 37) and WT (n = 43) infection were tested using a panel of 20 commercial antibody assays by seven manufacturers. Detailed information on the antibody assays, including test principle, target antigens, measuring units, covered immunoglobulin class, and cutoff values, are provided in Supplementary Table 1.
As shown in Figure 1, we observed significantly reduced detection rates in all commercial antibody assays based on S or RBD as target antigens with samples from convalescents after primary Omicron infection as compared to the WT control group (p < 0.05 for all anti-S and anti-RBD antibody assays; two-tailed Fisher’s exact test, Bonferroni correction for multiple testing).
In contrast, NC-based assays displayed no significant differences in the detection rates among convalescents after Omicron and WT infection (p > 0.05 in all assays).
Two IBLs, the SARS-CoV-2 ViraChip® IgG by Viramed and the recomLine SARS-CoV-2 IgG by Mikrogen, which integrate detection of both S-RBD and NC-specific antibodies into a single test result, were not affected by a reduced detection rate due to the unchanged sensitivity in measuring NC-specific antibodies.
The detailed results for all assays, including absolute and relative detection rates and comparative analyses, are displayed in Table 1.
Detection rates in subgroups of primarily Omicron infected convalescents
Next, we analyzed whether the detection rates of the commercial antibody assays were also reduced in the subgroup of convalescents, in whom a variant-specific PCR additionally confirmed Omicron infection. Omicron BA.1- or BA.2-specific PCR results were available for nine convalescents (BA.1: n = 4; BA.2: n = 5). As for the entire cohort (Figure 1), the detection rates were significantly reduced in almost all S- and RBD-specific immunoassays (13 of 16), while all NC-specific antibody assays displayed similar detection rates among Omicron or WT-infected groups (Supplementary Table 2).
To clarify whether the reduction in the detection rates of the assays was due to different antibody concentrations among the two groups, we calculated the diagnostic performances using only samples from Omicron and WT convalescents with NT titers ≥20 against the respective variant (Omicron: n = 30, WT n = 35). Indeed, the commercial antibody assays’ detection rates were also significantly reduced in samples with overall high antibody concentrations (all anti-S/anti-RBD assays p < 0.05; all anti-NC assays p > 0.05; Fisher’s exact test; Supplementary Table 3).
Correlation of quantitative antibody levels and variant-specific nAb titers
Finally, we analyzed the correlation between titers of variant-specific NT titers with antibody levels quantified by the commercial antibody assays. Figure 2 shows a robust correlation between RBD-ACE2 binding inhibition quantified by commercial sVNTs and the respective NT titers in convalescent samples obtained from WT-infected subjects (r = 0.7–0.8). In contrast, a much weaker correlation was observed in sera from Omicron-infected subjects (r = 0.2–0.3), as indicated by a flattened steepness of the regression line (Figure 2).
The S- and RBD-specific ELISAs, CLIAs, and the IBLs displayed an overall reduction in the signal intensity, i.e., the regression lines in the Omicron cohort shifted downwards (Figures 3a-3d, Supplementary Figures 2 - 8). In contrast to Anti-S- and Anti-RBD-immunoassays, the signal intensities and correlations were comparable for NC-specific antibody assays among both cohorts (Figures 3e and 3f, Supplementary Figures 2e, 3c, 5d, 7d, and 9).